There is an increased interest from the vaccine industry to use mammalian cell cultures for influenza vaccine manufacturing. Therefore, it became important to study the influenza infection mechanism, the viralhost interaction, and the replication kinetics from a bioprocessing stand point to maximize the influenza viral production yield in cell culture. In the present work, influenza replication kinetics was studied in HEK293 cells. Two infection conditions were evaluated, a low (0.01) and a high multiplicity of infection (1.0). Critical time points of the viral production cycle (infection, protein synthesis, viral assembly and budding, viral release, and host-cell death) were identified in small-scale cell cultures. Additionally, cell growth, viability, and viral titers were monitored in the viral production process. The infection state of the cultivated cell population was assessed by influenza immunolabeling throughout the culture period. Influenza virus production kinetics were also on-line monitored by dielectric spectroscopy and successfully correlated to real-time capacitance measures. Overall, this work provided insights into the mechanisms associated with the infection of human HEK293 cell line by the influenza virus and demonstrated, once again, the usefulness of multifrequency scanning permittivity for in-line monitoring and supervision of cell-based viral production processes. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 29: 275-284, 2013